PROJECT SUMMARY/ABSTRACT The increased incidence of autoimmune and inflammatory disorders highlights the need for improved therapeutics to treat these diseases. Our work places RIPK1 kinase at the apex of inflammatory pathways where it regulates a form of inflammatory cell death called necroptosis mediated by RIPK1, RIPK3 and MLKL. In the work proposed, we will delineate novel necroptosis pathways important in autoimmunity and immune-mediated liver injury operative in autoimmune and viral hepatitis. We demonstrate that mice with a dendritic cell RIPK1-deficiency (Ripk1DC KO) develop inflammation and autoimmunity; unexpectedly revealing that RIPK1 restrains necroptosis in dendritic cells (DC) in contrast to it well established role as an initiator of necroptosis. During development, RIPK1 prevents necroptosis mediated by the nucleic acid sensor Z-DNA binding protein (ZBP1) leading us to hypothesize that ZBP1- mediated dendritic cell necroptosis breaks tolerance and induces autoimmunity. We will test this hypothesis in Aim 1 by determining whether a ZBP1-deficiency prevents inflammation and autoimmunity in Ripk1DC KO mice. We will identify the nucleic acid species bound to ZBP1 and will establish whether nucleic acid binding is required to induce inflammation and/or autoimmunity. Nucleic acids in the Z-form are known to be highly immunogenic and anti-Z-DNA autoantibodies occur in lupus patients; therefore, we will test genetically whether inhibiting ZBP1 or necroptosis ameliorates disease in other autoimmune prone mice. In Aim 2, we will investigate whether RIPK1-kinase-dependent necroptosis contributes to a mouse model of autoimmune hepatitis (AIH) induced by the plant lectin Concanavalin A (Con A). Based on our preliminary data in reciprocal hematopoietic chimeras we hypothesize that AIH is mediated by canonical necroptosis in hematopoietic cells and noncanonical necroptosis in hepatocytes. Consistent with increased RIPK1 and MLKL expression in the livers of AIH patients, our preliminary data implicate RIPK1 and MLKL but not RIPK3 in hepatocyte cell death, leading us to hypothesize that a novel RIPK3 independent necroptosis pathway is induced in hepatocytes. Going forward we will test this hypothesis rigorously by generating mice that express kinase inactive RIPK1D138N or delete Ripk3 or Mlkl specifically in liver parenchymal cells. An additional goal will be to delineate this non-canonical necroptosis pathway, by identifying the RIPK1- interacting kinase responsible for MLKL activation in hepatocytes and to assess its contribution to AIH. Successful completion of these Aims will elucidate the contribution(s) of these new necroptosis pathways to autoimmunity and acute liver inflammation with the long-term goal to identify those autoinflammatory/immune diseases that might benefit from RIPK1 kinase targeted therapy